We propose a response to Challenge 06-AG-104 that incorporates development of three novel tools and technologies that can alter or interrogate human mitochondrial function in vivo. We will then apply these tools to the study of aging heart and muscle in order to demonstrate that they can enable an integrated understanding of the role of mitochondrial ROS, mitochondrial energetics and changes in mitochondrial protein composition and turnover in age-related sarcopenia and heart disease. In Aim 1 we will manipulate in vivo mitochondrial structure and activity by the use of novel mitochondrial targeted antioxidant and protective peptides. This is important in human aging because data from our and other laboratories indicate that mitochondria and mitochondrial ROS play a critical role in age-related declines in organ function. In Aim 2 we will apply novel magnetic resonance and optical spectroscopy techniques to measure O2 and ATP fluxes simultaneously to create a totally non-invasive and more versatile measure of mitochondrial function. In Aim 3 w e will use novel proteomic methodologies to characterize the structural basis of changes in mitochondrial function in aging tissues in vivo and tissues protected by antioxidant peptides. This will include measurement of global mitochondrial protein abundance and mitochondrial proteome-wide differences in protein syntheses and turnover rates using in vivo heavy isotope labeling. In each of these aims we will apply and validate the tools developed using two tissues for which there is abundant evidence for the critical role of mitochondria and, mitochondrial ROS in aging: heart and skeletal muscle. We will examine healthy young and old muscle, plus young hearts stressed with angiotensin II and young muscle stressed by AZT treatment. The combined use of these three methods allows us to manipulate human mitochondrial structure and activity at the same time as we monitor and measure mitochondrial function and dysfunction in aging tissues. Thus, we can obtain an integrated assessment of the in vivo inter-relationships of mitochondrial, ROS, energetics and proteomics in aging and disease. PUBLIC HEALTH RELEVANCE: This response to Challenge 06-AG-104 incorporates three responsive elements that will be used together in concert to provide an integrated assessment of the in vivo inter-relationships of mitochondrial, ROS, energetics and proteomics in aging and disease: 1) we will manipulate in vivo mitochondrial structure and activity by the use of novel mitochondrial targeted antioxidant and protective peptides;2) we will use novel magnetic resonance and optical spectroscopy tools to create totally non-invasive and more versatile measures of mitochondrial energetics in vivo, and 3) we will use novel proteomic tools to characterize the structural basis of changes in mitochondrial function. The combined use of these methods allows us to manipulate human mitochondrial structure and activity at the same time as we monitor and measure mitochondrial function and dysfunction in aging tissues.